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α-1,2 Linkages in dextran NRRL B 1424
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
a-1 ,2 Linkages MITSUO
TORII,
ELVIN
103, 283-285 (1963)
in Dextran A. KABAT,
AND
NRRL B 1424’ SHERMAN
BEYCHOK
From the Departments of Microbiology, Neurology and Biochemistry, College of Physicians and Surgeons, Columbia University; and the Neurological Institute, Presbyterian Hospital, New Fork Received
June 13, 1963
Immunochemical evidence for the presence of a-1,2 linkages in dextran B 1424 has been confirmed by acetolysis and isolation of kojibiose.
Immunochemical evidence has accumulated indicat#ing that dextran NRRL B 1424 (1) should contain a considerable proportion of a-l,2 linked glucoses. This dextran reacts strongly with human antidextran sera with specificities involving (~-1~2 linked glucosyl residues (2) and cross reacts with type XII antipneumococcal serum (3) about as strongly as do dextrans known, from optical rotation studies of the cuprammonium complexes (4) and by the isolation of kojibiose (a-glucosyl-1,2-glucose) (5, 6)) to contain a-l,2 linked glucose residues. Moreover, rabbit and human anti-1424 sera reacted most strongly with the dextrans with high contents of a-l,2 linkages, and kojibiose was the best disaccharide inhibitor of the 1424-anti-1424 reaction (7). Since chemical data on the presence of a-l,2 linked glucoses in this dextran are not available, it was considered desirable to attempt the isolation of kojibiose from this dextran. a-l ,2 and a-l ,3 linkages have been found to be less stable than a-l,6 linkages to acid hydrolysis, but more stable to acetolysis (8). This latter procedure had been used to isolate kojibiose (5, 6) and nigerose (9., 6, 10) from various dextrans. Accordingly, a sample2 of dextran B 1424 1 Aided by Grants from the National Science Foundation (G 18727), the Office of Naval Research, and t,he General Research Support Grant and GM-10576-01 from the National Institutes of Health, USPHS. 2 Kindly supplied by Dr. Allene Jeanes, Northern Regional Utilization Laboratory, Peoria, Illinois. 283
NRRL
was acetolyzed according to the procedure of Matsuda et al. (10). For comparison, dextran B-l299-S-32 from which kojibiose had also been isolated (5, 6) was acetolyzed in the same manner. To 43.3 mg. dextran B 1424, 0.37 ml. of a solution prepared by mixing 4.8 ml. acetic anhydride, 3.2 ml. glacial acetic acid, and 0.6 ml. concentrated H&04, was added. After 7 days at room temperature and 30 minutes at 8O”C., ice water was added, the solution neutralized with Na2COB, and allowed to stand overnight at 4°C. It was then extracted with chloroform, the chloroform extract washed with water, and dried over anhydrous Na2S04. The dry chloroform extract was concentrated to dryness in vacua, the residue dissolved in 0.8 ml. dry methanol, and 0.045 ml. of N/l sodium methoxide was added. After standing overnight at 4”C., a small quantity of water was added and Na+ was removed with Amberlite IR-120 (H). The solution was concentrated to a syrup in vacua and taken up in water. Descending paper chromatography on S & S 589 for 16 hr. in n-propanol-ethyl acetate-water (6 : 1: 3 v/v) and development wit’h alkaline silver nitrate showed three major spots corresponding to glucose, kojibiose, and isomaltose. A fraction corresponding in Rf to kojibiose was eluted from the paper and lyophilized (8.2 mg.); the parallel experiment with dextran 1299 yielded 11.3 mg. from 43.3 mg. dextran. The isolated fraction from 1424 gave a major spot corresponding to kojibiose and a faint spot corresponding to isomaltose. The specific rotation [a];:; was
284
TORII,
KABAT,
AND
0 .
FIG. 1. Variation
Acefyfated Acetylated
of specific rotation
+ 116” while that from B 1299 was + 118”. Authentic a-kojibiose has been reported to give [&? = +162” -+ 137” (17 hr.). Reducing sugar equivalents of the isolated compounds as glucose by the Park-Johnson method were as follows: from B 1424, 22 %; from B 1299,19 %; authentic kojibiose, 18 %. The lower optical rotation and the slightly higher glucose value were probably due to the slight contamination with isomaltose. For further identification the acetates were prepared: the lyophilized material, 7.0 mg., was treated with 0.085 ml. of acetic anhydride-pyridine (2:3 v/v) at 0°C. for 3 days. On adding 0.25 ml. of ice water, an oil separated which solidified. It was washed with a few drops of water and dried (11.8 mg.). Recrystallization from 95 % ethanol gave 4.0 mg. of crystals, m. p. 166167°C.; a mixed melting point with authentic a-kojibiose octaacetate (m. p. 166-168°C.) (Yamauchi and Aso (II), 166°C.) showed no depression. With B 1299, 5.8 mg. acetate were obtained from 9.8 mg. lyophilized material, m. p. 166168°C.; mixed melting point with authentic kojibiose octaacetate showed no depression. Optical rotatory dispersion curves of the acetylated disaccharides were measured using a Bendix-Ericsson automatic record-
BEYCHOK
from
with wavelength.
ing spectropolarimeter over the wavelength range 589-250 mp. The concentrations of the acetylated disaccharide from B 1299 and of authentic acetylated kojibiose were both 0.070%, and the observed rotations could not be distinguished at any wave length; that of the acetylated disaccharide from B 1424 was 0.073 %. When converted to specific rotations, (Fig. l), the values obtained for authentic acetylated kojibiose and the corresponding product from B 1424 were indistinguishable, within the experimental error, over the entire range. At the lower wavelengths (350-250 mp), where the greatest differences would be anticipated for nonidentical samples, point by point comparison revealed agreement better than 1%. Further analysis of the data (cf. 13) indicated that the dispersion of the samples obeyed a single-term Drude equation over the entire wavelength interval with a dispersion constant, X,, of 160 f 2 mp. Acetolysis has shown (9, 10) not to cause significant reversion of glucose to kojibiose or nigerose; this was confirmed in a control experiment. Acid hydrolysis, however, is known to cause some reversion (12). REFERENCES 1. JEANES, A., HAYNES, W. C., WILHBM, C. A., RANKIN, S. C., MELVIN, E. H., AUSTIN,
a-1,2 M.
J.,
LINKAGES
IN DEXTRAN
J. E., FISHER, B. E., H. M., AND RIST, C. E., J. Am. Chem. Sot. 76, 5041 (1954). ALLEN, P. Z., AND KBBAT, E. A., J. Am. Chem. Sot. 81, 4382 (1959). GOODMAN, J. W., AND KABAT, E. A., J. Immunol. 84, 347 (1960). SCOTT, T. A., HELLMAN, N. N., AND SENTI, F., J. Am. Chem. Sot. 79, 1178 (1957). MATSUDA, K., FUJIMOTO, K., AND Aso, K., Tohoku J. Agr. Res. 12, 359 (1961). M.ITSUDA, K., W.~TANABE, H., FUJIMOTO, K., AND Aso, K., Nature 191, 278 (1961). MAGE, R. G., .ZND K.isa~, E. A., J. Immunol. 91, (1963). C~LUSKEY,
3. 4.
5. 6.
7.
9.
10. 11. 12.
13.
B 1424
285
K., MATSUDA, K., AND Aso, K., Tohoku J. Agr. Res. 13. 61 (1962). GOLDSTEIN, I. S., AND WHEL.IN, W. J., cited W. J., Bull. Sot. Chim. Biol. by WHELAN, 42, 1569 (1960); J. Chem. Sot., p. 170 (1962). MATSUDA, K., WAT.~NABE, H., AND Aso, K., Tohoku J. Agr. Res. 12, 351 (1961). YAMAUCHI, F., AND Aso, K., Kature 189, 753 (1961). THOMPSON, A., ANNO, K., WOLFROM, M. L., AND INBTOME, M., J. Am. Chem. Sot. 76, 1309 (1954). SCHELLMAN, J., Comp. Rend. Trav. Lab. Curlsberg 30, 363 (1958).
8. FUJIMOTO,
TSUCHIYA,
2.
NRRL
OF
BIOCHEMISTRY
AND
BIOPHYSICS
a-1 ,2 Linkages MITSUO
TORII,
ELVIN
103, 283-285 (1963)
in Dextran A. KABAT,
AND
NRRL B 1424’ SHERMAN
BEYCHOK
From the Departments of Microbiology, Neurology and Biochemistry, College of Physicians and Surgeons, Columbia University; and the Neurological Institute, Presbyterian Hospital, New Fork Received
June 13, 1963
Immunochemical evidence for the presence of a-1,2 linkages in dextran B 1424 has been confirmed by acetolysis and isolation of kojibiose.
Immunochemical evidence has accumulated indicat#ing that dextran NRRL B 1424 (1) should contain a considerable proportion of a-l,2 linked glucoses. This dextran reacts strongly with human antidextran sera with specificities involving (~-1~2 linked glucosyl residues (2) and cross reacts with type XII antipneumococcal serum (3) about as strongly as do dextrans known, from optical rotation studies of the cuprammonium complexes (4) and by the isolation of kojibiose (a-glucosyl-1,2-glucose) (5, 6)) to contain a-l,2 linked glucose residues. Moreover, rabbit and human anti-1424 sera reacted most strongly with the dextrans with high contents of a-l,2 linkages, and kojibiose was the best disaccharide inhibitor of the 1424-anti-1424 reaction (7). Since chemical data on the presence of a-l,2 linked glucoses in this dextran are not available, it was considered desirable to attempt the isolation of kojibiose from this dextran. a-l ,2 and a-l ,3 linkages have been found to be less stable than a-l,6 linkages to acid hydrolysis, but more stable to acetolysis (8). This latter procedure had been used to isolate kojibiose (5, 6) and nigerose (9., 6, 10) from various dextrans. Accordingly, a sample2 of dextran B 1424 1 Aided by Grants from the National Science Foundation (G 18727), the Office of Naval Research, and t,he General Research Support Grant and GM-10576-01 from the National Institutes of Health, USPHS. 2 Kindly supplied by Dr. Allene Jeanes, Northern Regional Utilization Laboratory, Peoria, Illinois. 283
NRRL
was acetolyzed according to the procedure of Matsuda et al. (10). For comparison, dextran B-l299-S-32 from which kojibiose had also been isolated (5, 6) was acetolyzed in the same manner. To 43.3 mg. dextran B 1424, 0.37 ml. of a solution prepared by mixing 4.8 ml. acetic anhydride, 3.2 ml. glacial acetic acid, and 0.6 ml. concentrated H&04, was added. After 7 days at room temperature and 30 minutes at 8O”C., ice water was added, the solution neutralized with Na2COB, and allowed to stand overnight at 4°C. It was then extracted with chloroform, the chloroform extract washed with water, and dried over anhydrous Na2S04. The dry chloroform extract was concentrated to dryness in vacua, the residue dissolved in 0.8 ml. dry methanol, and 0.045 ml. of N/l sodium methoxide was added. After standing overnight at 4”C., a small quantity of water was added and Na+ was removed with Amberlite IR-120 (H). The solution was concentrated to a syrup in vacua and taken up in water. Descending paper chromatography on S & S 589 for 16 hr. in n-propanol-ethyl acetate-water (6 : 1: 3 v/v) and development wit’h alkaline silver nitrate showed three major spots corresponding to glucose, kojibiose, and isomaltose. A fraction corresponding in Rf to kojibiose was eluted from the paper and lyophilized (8.2 mg.); the parallel experiment with dextran 1299 yielded 11.3 mg. from 43.3 mg. dextran. The isolated fraction from 1424 gave a major spot corresponding to kojibiose and a faint spot corresponding to isomaltose. The specific rotation [a];:; was
284
TORII,
KABAT,
AND
0 .
FIG. 1. Variation
Acefyfated Acetylated
of specific rotation
+ 116” while that from B 1299 was + 118”. Authentic a-kojibiose has been reported to give [&? = +162” -+ 137” (17 hr.). Reducing sugar equivalents of the isolated compounds as glucose by the Park-Johnson method were as follows: from B 1424, 22 %; from B 1299,19 %; authentic kojibiose, 18 %. The lower optical rotation and the slightly higher glucose value were probably due to the slight contamination with isomaltose. For further identification the acetates were prepared: the lyophilized material, 7.0 mg., was treated with 0.085 ml. of acetic anhydride-pyridine (2:3 v/v) at 0°C. for 3 days. On adding 0.25 ml. of ice water, an oil separated which solidified. It was washed with a few drops of water and dried (11.8 mg.). Recrystallization from 95 % ethanol gave 4.0 mg. of crystals, m. p. 166167°C.; a mixed melting point with authentic a-kojibiose octaacetate (m. p. 166-168°C.) (Yamauchi and Aso (II), 166°C.) showed no depression. With B 1299, 5.8 mg. acetate were obtained from 9.8 mg. lyophilized material, m. p. 166168°C.; mixed melting point with authentic kojibiose octaacetate showed no depression. Optical rotatory dispersion curves of the acetylated disaccharides were measured using a Bendix-Ericsson automatic record-
BEYCHOK
from
with wavelength.
ing spectropolarimeter over the wavelength range 589-250 mp. The concentrations of the acetylated disaccharide from B 1299 and of authentic acetylated kojibiose were both 0.070%, and the observed rotations could not be distinguished at any wave length; that of the acetylated disaccharide from B 1424 was 0.073 %. When converted to specific rotations, (Fig. l), the values obtained for authentic acetylated kojibiose and the corresponding product from B 1424 were indistinguishable, within the experimental error, over the entire range. At the lower wavelengths (350-250 mp), where the greatest differences would be anticipated for nonidentical samples, point by point comparison revealed agreement better than 1%. Further analysis of the data (cf. 13) indicated that the dispersion of the samples obeyed a single-term Drude equation over the entire wavelength interval with a dispersion constant, X,, of 160 f 2 mp. Acetolysis has shown (9, 10) not to cause significant reversion of glucose to kojibiose or nigerose; this was confirmed in a control experiment. Acid hydrolysis, however, is known to cause some reversion (12). REFERENCES 1. JEANES, A., HAYNES, W. C., WILHBM, C. A., RANKIN, S. C., MELVIN, E. H., AUSTIN,
a-1,2 M.
J.,
LINKAGES
IN DEXTRAN
J. E., FISHER, B. E., H. M., AND RIST, C. E., J. Am. Chem. Sot. 76, 5041 (1954). ALLEN, P. Z., AND KBBAT, E. A., J. Am. Chem. Sot. 81, 4382 (1959). GOODMAN, J. W., AND KABAT, E. A., J. Immunol. 84, 347 (1960). SCOTT, T. A., HELLMAN, N. N., AND SENTI, F., J. Am. Chem. Sot. 79, 1178 (1957). MATSUDA, K., FUJIMOTO, K., AND Aso, K., Tohoku J. Agr. Res. 12, 359 (1961). M.ITSUDA, K., W.~TANABE, H., FUJIMOTO, K., AND Aso, K., Nature 191, 278 (1961). MAGE, R. G., .ZND K.isa~, E. A., J. Immunol. 91, (1963). C~LUSKEY,
3. 4.
5. 6.
7.
9.
10. 11. 12.
13.
B 1424
285
K., MATSUDA, K., AND Aso, K., Tohoku J. Agr. Res. 13. 61 (1962). GOLDSTEIN, I. S., AND WHEL.IN, W. J., cited W. J., Bull. Sot. Chim. Biol. by WHELAN, 42, 1569 (1960); J. Chem. Sot., p. 170 (1962). MATSUDA, K., WAT.~NABE, H., AND Aso, K., Tohoku J. Agr. Res. 12, 351 (1961). YAMAUCHI, F., AND Aso, K., Kature 189, 753 (1961). THOMPSON, A., ANNO, K., WOLFROM, M. L., AND INBTOME, M., J. Am. Chem. Sot. 76, 1309 (1954). SCHELLMAN, J., Comp. Rend. Trav. Lab. Curlsberg 30, 363 (1958).
8. FUJIMOTO,
TSUCHIYA,
2.
NRRL